Membrane-targeting Properties Research Articles

Implication of membrane localization of target mRNA in the action of a small RNA: mechanism of post-transcriptional regulation of glucose transporter in Escherichia coli

Accumulation of phosphosugars such as glucose-6-phosphate causes a rapid degradation of ptsG mRNA encoding the major glucose transporter IICB(Glc) in an RNase E/degradosome-dependent manner. The destabilization of ptsG mRNA is caused by a small antisense RNA (SgrS) that is induced by phosphosugar stress. In this study, we analyzed a series of ptsG-crp translational fusions to identify the mRNA region required for the rapid degradation of ptsG mRNA. We found that the ptsG-crp mRNA is destabilized in response to phosphosugar stress when it contains the 5' portion of ptsG mRNA corresponding up to the first two transmembrane domains (TM1 and TM2) of IICB(Glc). The destabilization of ptsG-crp mRNA was largely eliminated by frameshift mutations in the transmembrane region. The IICB(Glc)-CRP fusion proteins containing more than two transmembrane domains were localized at the membrane. The efficient destabilization of ptsG-crp mRNA was restored when TM1 and TM2 of IICB(Glc) were replaced by part of the LacY transmembrane region. We conclude that the membrane-targeting property of IICB(Glc) protein rather than the particular nucleotide or amino acid sequence is required for the efficient degradation of ptsG mRNA in response to metabolic stress. The stimulation of ptsG-crp mRNA degradation was completely eliminated when either the hfq or sgrS gene is inactivated. The efficient mRNA destabilization was observed in the absence of membrane localization when translation was reduced by introducing a mutation in the ribosome-binding site in the cytoplasmic ptsG-crp mRNA. Taken together, we conclude that mRNA localization to the inner membrane coupled with the membrane insertion of nascent peptide mediates the Hfq/SgrS-dependent ptsG mRNA destabilization presumably by reducing second rounds of translation.

Link between genome packaging and rate of budding for Rous sarcoma virus.

The subcellular location at which genomic RNA is packaged by Gag proteins during retrovirus assembly remains unknown. Since the membrane-binding (M) domain is most critical for targeting Gag to the plasma membrane, changes to this determinant might alter the path taken through the cell and reduce the efficiency of genome packaging. In this report, a Rous sarcoma virus (RSV) mutant having two acidic-to-basic substitutions in the M domain is described. This mutant, designated Super M, produced particles much faster than the wild type, but the mutant virions were noninfectious and contained only 1/10 the amount of genomic RNA found in wild-type particles. To identify the cause(s) of these defects, we considered data that suggest that RSV Gag traffics through the nucleus to package the viral genome. Although inhibition of the CRM-1 pathway of nuclear export caused the accumulation of wild-type Gag in the nucleus, nuclear accumulation did not occur with Super M. The importance of the nucleocapsid (NC) domain in membrane targeting was also determined, and, importantly, deletion of the NC sequence prevented plasma membrane localization by wild-type Gag but not by Super M Gag. Based on these results, we reasoned that the enhanced membrane-targeting properties of Super M inhibit genome packaging. Consistent with this interpretation, substitutions that reestablished the wild-type number of basic and acidic residues in the Super M Gag M domain reduced the budding efficiency and restored genome packaging and infectivity. Therefore, these data suggest that Gag targeting and genome packaging are normally linked to ensure that RSV particles contain viral RNA.

8] Molecular structure of membrane-targeting calcium sensors in vision: Recoverin and guanylate cyclase-activating protein 2

Publisher Summary An important class of calcium-binding proteins present in retinal photoreceptor cells includes recoverin from mammalian rods, guanylate cyclase-activating proteins (GCAP-1, GCAP-2, and GCAP-3) from mammalian rods and cones, and guanylate cyclase inhibitory protein (GCIP) from frog rods. The recoverin branch of the EF-hand superfamily includes neuronal Ca 2+ sensors such as neurocalcin, frequenin, and hippocalcin. Mass spectrometric analysis of retinal recoverin and the GCAP proteins revealed that they are myristoylated at the amino terminus. GCAP-2 exhibits different membrane-targeting properties from those of recoverin. The myristoylated and unmyristoylated forms of GCAP-2 bind to membranes at low Ca 2+ concentrations ( 50 mM NaCl).This chapter presents a detailed structural analysis of recoverin and GCAP-2. The Ca 2+ -induced structural changes in these proteins are important for elucidating their membrane-targeting mechanisms and for understanding the molecular mechanism of Ca 2+ -sensitive regulation of phototransduction.

Structural requirements for the cytotoxicity of the N-terminal region of HIV type 1 Nef.

We have found that the hemolytic and cytotoxic activities of myristoylated Nef N-terminal peptides require a net positive charge in the first seven amino residues of the sequence. The activities are considerably less dependent on the secondary structure of the peptides. Film balance studies showed that both active and inactive peptides interacted with neutral phospholipid monolayers, suggesting that binding to neutral lipids was not a sufficient condition for lytic activity. It was also found that nonmyristoylated N-terminal peptide did not interact to the same extent with the monolayer, indicating that myristoylation was essential for lipid interaction. It is considered that the positively charged residues of the proximate N terminus of Nef interact with acidic lipids of biological membranes, reinforcing the weak membrane-targeting properties of the myristyl chain. Parallels are drawn between this mode of interaction with membranes and that of members of the Src family of proteins, which are also myristoylated and have positively charged residues in their proximate N termini. In particular, these proteins and Nef also have serine residues in their proximal N-terminal regions, which when phosphorylated could neutralize the positive charge and thus provide a mechanism for modulating membrane interaction.

Distinct Functional Properties of Rab3A and Rab3B in PC12 Neuroendocrine Cells

Rab3A and Rab3B are highly homologous monomeric GTPases that are putative regulators of exocytosis in those tissues in which they are expressed. We have characterized and directly compared the targeting and functional properties of these isoforms in PC12 neuroendocrine cells. Rab3A and Rab3B both targeted to norepinephrine (NE)-containing large dense core vesicles (LDCVs) when stably expressed in PC12 cells, as determined by immunofluorescence and membrane fractionation. Both Rab3 isoforms also bound to recombinant rabphilin-3A in a GTP-dependent manner. The membrane association of rabphilin-3A was modestly enhanced in Rab3B-expressing PC12 cells relative to Rab3A-overexpressing cells. In addition, overexpression of Rab3A modestly inhibited Ca2+-evoked NE release, whereas Rab3B and a GTP binding mutant (Rab3B N135I) markedly stimulated the efficiency of [3H]NE secretion by PC12 cells (i.e. secretion normalized to total cell radioactivity). Expression of Rab3B and Rab3B N135I increased not only the efficiency of NE secretion but also the accumulation of [3H]NE into LDCVs (i.e. the secretory cargo available for secretion). Neither of these effects was attributable to changes in the numbers of LDCVs nor the docking of LDCVs at the plasma membrane. Our results indicate that Rab3A and Rab3B have similar membrane targeting properties and are capable of interacting with the same putative downstream effector; i.e. rabphilin-3A. However, these isoforms are functionally distinct monomeric GTPases with Rab3B stimulating a late step in Ca2+-evoked secretion when expressed in PC12 cells.